Chitinases are produced throughout the growth process of fungi and are thought to play important roles in morphogenesis. Aspergillus fumigatus, is an important pathogen of immunocompromised individuals in which it causes pneumonia and invasive disseminated disease with high mortality; it is also known to produce chitinase. We have induced an exceptionally stable extracellular chitinase in A. fumigatus YJ-407, which could be isolated readily in a homogeneous form by using ammonium sulfate precipitation followed by DEAE-cellulose chromatography and preparative PAGE. The molecular mass of this chitinase was estimated to be 46 000 by SDS/PAGE, and its isoelectric point was pH 5.6. The enzyme was most active at pH 5.0 and 60 8C, and was inhibited strongly by Hg . The enzyme was stable over a broad pH range 4±8 and below 45 8C. Tryptophan and carboxyl groups were found to be essential for the enzyme activity. The Michaelis constants for swollen chitin and chitosan were 1.12 mg´mL 21 and 1.84 mg´mL 21 , respectively. The enzyme showed maximum activity towards glycol chitin and partially deacetylated chitosan, and lower activity towards colloidal chitin. Analysis of the hydrolysis product showed that the enzyme has both endoand exo-hydrolytic activities. In addition, a transglycosyl activity was also observed.Keywords: Aspergillus fumigatus; chitinase; endochitinase; exochitinase; transglycosylation.Chitinases, enzymes that cleave the bond between the C1 and C4 of two consecutive N-acetylglucosamines of chitin, have been found in microorganisms, plants and animal tissues. These enzymes have been shown to have a variety of functions. In plants, which lack chitin, chitinases are thought to be a defence system against fungal pathogens. Chitinases produced by bacteria appear to have a nutritional or scavenging role, while those produced by filamentous fungi have been shown to be involved in a variety of functions such as cell wall digestion, germination of spores, hyphal growth, hyphal autolysis, differentiation into spores, assimilation of chitin and mycoparasitism [1±3]. However, their roles in fungal growth and mechanisms of chitinase regulation are almost totally unknown.As one of the most ubiquitous of the airborne saprophytic fungi, Aspergillus fumigatus has been shown to be an opportunistic pathogen causing pneumonia and other fatal invasive infections in immunocompromised hosts, particularly among patients undergoing cytotoxin chemotherapy or bone-marrow transplantation [4±6]. There has been a dramatic increase in severe and usually fatal invasive aspergillosis caused by A. fumigatus. This organism is also known to secrete extracellular chitinases [7,8]. To understand the pathogenesis associated with A. fumigatus infection, we have studied the chitinases in this organism. An extracellular chitinase, which was remarkably resistant to physical and chemical manipulations, was induced from A. fumigatus YJ-407 and could be isolated readily in homogeneous form by ammonium sulfate precipitation followed by DEAE-cellulose c...
Phytophthora root rot (PRR) of soybean (Glycine max (L.) Merr.) is the second most important cause of yield loss by disease in North America, surpassed only by soybean cyst nematode (Wrather et al. in Can J Plant Pathol 23:115-121, 2001). Tolerance can provide economically useful disease control, conditioning partial resistance of soybean to PRR. The aims of this study were to identify new quantitative trait loci (QTL) underlying tolerance to PRR, and to evaluate the effects of pyramided or stacked loci on the level of tolerance. A North American cultivar 'Conrad' (tolerant to PRR) was crossed with a northeastern China cultivar 'Hefeng 25' (tolerant to PRR). Through single-seed descent, 140 F2:5 and F2:6 recombinant inbred lines were advanced. A total of 164 simple sequence repeat (SSR) markers were used to construct a genetic linkage map. The percentage of seedling death was measured over 2 years (2007 and 2008) in the field at four naturally infested locations in Canada and China following additional soil infestation and in the greenhouse following inoculation with Phytophthora sojae isolate. A total of eight QTL underlying tolerance to PRR were identified, located in five linkage groups (F, D1b+w, A2, B1, and C2). The phenotypic variation contributed by the loci ranged from 4.24 to 27.98%. QPRR-1 (anchored in the interval of SSR markers Satt325 and Satt343 of LG F), QPRR-2 (anchored in the interval of Satt005 and Satt600 of LG D1b+w), and QPRR-3 (anchored in the interval of Satt579 and Sat_089 of LG D1b+w) derived their beneficial allele from 'Conrad'. They were located at chromosomal locations known to underlie PRR tolerance in diverse germplasm. Five QTL that derived beneficial alleles from 'Hefeng 25' were identified. The QTL (QPRR-1 to QPRR-7) that were detected across at least three environments were selected for loci stacking and to analyze the relationship between number of tolerance loci and disease loss percentage. The accumulation of tolerance loci was positively correlated with decreases in disease loss percentage. The pyramid of loci underlying tolerance to PRR provided germplasm useful for crop improvement by marker-assisted selection and may provide durable cultivar tolerance against the PRR disease.
A method has been developed for the separation and determination of phospholipids by nonaqueous capillary electrophoresis in a separation medium of acetonitrile-2-proponol (3:2, v/v), 0.3% acetic acid and 60 mM ammonium acetate. To optimize the separation conditions, the composition of separation medium including alcohols, acetic acid, n-hexane and ammonium acetate was studied. The solvation interaction and ion-dipole interaction were also investigated. The contents of phospholipids in soybean, sunflower, peanut, apricot kernel, filbert and walnut were determined by the recommended method. The results obtained by the nonaqueous capillary electrophoreses were in good agreement with those determined by micellar electrokinetic chromatography.
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